Summary of work: Genotoxic/oxidative stresses contribute to the development of degenerative diseases, and may underlie the aging process itself. Cells respond to such stresses with the induction of numerous gene products, but much remains to be learned concerning the signaling pathways mediating these effects or the functional significance of the induced gene products. This project encompasses studies related to these cellular responses. Efforts over the past year have been focused in several areas: 1) Activation of Akt Kinase by Oxidative Stress. We have demonstrated that hydrogen peroxide treatment results in the activation of a downstream mediator of PI-3 kinase signaling, Akt, in a variety of human cell types. Recent findings from several laboratories have implicated the PI-3 kinase/Akt pathway in enhancing survival in response to growth factor deprivation. In keeping with this view we found that inhibitors of PI-3 kinase (i.e. wortmannin) likewise prevented activation of Akt by hydrogen peroxide and enhanced the cytotoxicity seen in hydrogen peroxide-treated cells. These findings suggest an important survival role for this pathway during the cellular response to oxidant injury. 2) Role of Posttranscriptional Events in the Reglation of Cyclin D1 Expression Following Stress. Recent studies from our laboratory have demonstrated that cyclin D1 expression is greatly reduced following treatment with many stressful agents including the cyclopentenone prostaglandin PGA2. We have begun to explore the mechanism contributing to this down-regulation and have found that this occurs largely through posttranscriptional events leading to reduced stability of cyclin D1 mRNA. Actinomycin D treatment can prevent this loss, suggesting that a short-lived stress-inducible destabilizing protein is involved. Current studies are aimed at identifying this protein, and the mechanism through which it acts. 3) Role of c-Jun in Influencing Cell Survival Following Stress. We are employing cell lines harboring a dominant negative mutant form of c-jun (which can not be phosphorylated and activated) to test the role of this transcription factor in influencing cell surival in the gliobastoma cell line T98G following different stresses. Our results indicate that inhibition of c-jun activity sensitizes cells to DNA damaging stresses (UVC irradiation, x-irradiation, etoposide) but does not affect sensitivity to other non-DNA damaging stresses that induce apoptosis (tunicamycin, thapsigargin, taxol). Current studies are exploring the basis for this differential sensitivity.